The present invention relates to synthetic substrates which are to be used as reagents for the assay of various mammalian enzymes. More particularly, it relates to synthetic substrates containing a radiolabel which are especially useful in radioassays for proteolytic and peptidase enzymes.
Each of the substrates according to the invention is especially suitable for quantitative determination of one or more of the enzymes discussed herein. Further, each of the substrates can be used for a study of reactions in which an enzyme is formed, inhibited or consumed, or for determination of factors influencing or taking part in such a reaction. Synthetic substrates for enzyme determination have great advantages as compared to the natural ones, provided that they fulfill certain conditions, such as affinity for the enzyme, sensitivity and specificity for the enzyme, solubility in water or other biological test liquid and easy detectability of at least one of the remnants obtained upon hydrolytic cleavage.
The following abbreviations are used herein:
Ala alanine PA1 Aoc amyloxycarbonyl PA1 Arg arginine PA1 Asn asparagine PA1 Asp aspartic acid PA1 Aze 2-azetidine carboxylic acid PA1 Boc t-butyloxycarbonyl PA1 Bz benzoyl PA1 cpc cyclopentanecarbonyl PA1 Cys cysteine PA1 Dns dansyl PA1 Gln glutamine PA1 Glu glutamic acid PA1 &lt;Glu pyroglutamic acid PA1 Gly glycine PA1 His histidine PA1 Ile isoleucine PA1 Leu leucine PA1 Lys lysine PA1 Met methionine PA1 MNS 4-methoxy-2-naphthyl-amide PA1 Nle norleucine PA1 Orn ornithine PA1 Phe phenylalanine PA1 Sar sarcosine PA1 Pip pipecolic acid PA1 Pro proline PA1 .DELTA.Pro 3,4-dehydroproline PA1 Ser serine PA1 Thr threonine PA1 Trp tryptophan PA1 Tyr tyrosine PA1 Val valine PA1 Z benzyloxycarbonyl PA1 Cbo.dbd.CbO carbobenzoxy PA1 Tyr-Y PA1 Met-Y PA1 Arg-Y PA1 &lt;Glu-Y PA1 Leu-Y PA1 Phe-Y PA1 Trp-Y PA1 Ile-Y PA1 Val-Y PA1 Ala-Y PA1 Pro-Y PA1 Cys-Y PA1 His-Y PA1 S-(Bz)-Cys-Y PA1 Leu-Y PA1 X-Gly-Phe PA1 X-Gly-Arg PA1 X-Ala-Phe PA1 X-Ala-Arg PA1 X-Phe-Phe PA1 X-Phe-Arg PA1 X-Gly-Trp PA1 X-Gly-Tyr PA1 X-Gly-Ala PA1 X-Gly-Lys PA1 X-Ala-Trp PA1 X-Ala-Tyr PA1 X-Ala-Ala PA1 X-Ala-Lys PA1 X-Phe-Tyr PA1 X-Phe-Trp PA1 X-Phe-Ala PA1 X-Tyr-Tyr PA1 X-Trp-Tyr PA1 X-Tyr-Trp PA1 X-Trp-Trp PA1 X-Tyr-Ala PA1 X-Trp-Ala PA1 X-Phe-Lys PA1 X-Trp-Lys PA1 X-Tyr-Lys PA1 X-Gly-Phe PA1 glutaryl-Phe-Y PA1 succinoyl-Ala-Ala-Pro-Phe-Y PA1 acetyl-Trp-Y PA1 Ala-Ala-Phe-Y PA1 R-Bz-Phe-Y PA1 R-Bz-Phe-Gly-Gly-Y PA1 R-Bz-Phe-Gly-Y PA1 N-(Bz)-Tyr-Y PA1 R-(N-acetyl)-Phe-Y PA1 N-(Bz)-Trp-Y PA1 R-Tyr-Y PA1 .alpha.-Bz-Lys-Y PA1 acetyl-Trp-Gly-Y PA1 acetyl-Trp-Gly-Gly-Y PA1 acetyl-Trp-Ala-Y PA1 Acetyl-Trp-Ala-Gly-Y PA1 Bz-Tyr-Phe-Y PA1 Bz-Phe-Try-Y PA1 Bz-Tyr-Tyr-Y PA1 Z-Arg-Y PA1 Bz-Arg-Y PA1 (p-R)-Bz-Phe-Val-Arg-Y PA1 N-(acetyl)-Phe-Val-Arg-Y PA1 cyclohexylcarbonyl-Phe-Val-Arg-Y PA1 N-(tosyl)-Phe-Val-Arg-Y PA1 N-(p-aminobenzoyl)-Phe-Val-Arg-Y PA1 Phe-Val-Arg-Y PA1 Bz-Phe-Val-Arg-Y PA1 Phe-Val-Arg-Y PA1 Bz-(D)Phe-Val-Arg-Y PA1 Tyr-Val-Arg-Y PA1 N-(B)-Tyr-Val-Arg-Y PA1 (4-aminocyclohexylcarbonyl)-Phe-Val-Arg-Y PA1 (4-amino butyryl)-Phe-Val-Arg-Y PA1 2-(4-amino phenyl)acetyl-Phe-Val-Arg-Y PA1 N-(Bz)-Phe-Val-Lys-Y PA1 N-(Bz)-Leu-Leu-Arg-Y PA1 .beta.-cyclohexyl-Ala-Val-Arg-Y PA1 N-(Bz)-.beta.-cyclohexyl-Ala-Val-Arg-Y PA1 N-(Bz)(N-cyclohexyl)-.beta.-Ala-Val-Arg-Y PA1 N-(Bz)-Val-Arg-Y PA1 N-(Bz)-Val-Val-Arg-Y PA1 N-(Bz)-Leu-Val-Arg-Y PA1 N-(Bz)-Ile-Val-Arg-Y PA1 N-(Bz)-Val-Ile-Arg-Y PA1 N-(Bz)-Ile-Ile-Arg-Y PA1 N-(Bz)-Leu-Ile-Arg-Y PA1 Cbo-Arg-Arg-Arg-Y PA1 &lt;Glu-Gly-Arg-Y PA1 Bz-Ile-Glu-Gly-Arg-Y PA1 (D)Ala-Ala-Arg-Y PA1 (D)Leu-Gly-Arg-Y PA1 (D)Leu-Ile-Arg-Y PA1 (D)Leu-Val-Arg-Y PA1 (D)Leu-Val-Lys-Y PA1 (D)Pip-Phe-Arg-Y PA1 (D)Leu-Leu-Arg-Y PA1 Z-Ala-Arg-Arg-Y PA1 Bz-Phe-Val-Arg-Y PA1 sarcosyl-Pro-Arg-Y PA1 tosyl-Gly-Pro-Arg-Y PA1 (D)Phe-Pip-Arg-Y PA1 (D)Phe-Pro-Arg-Y PA1 Val-Pro-Arg-Y PA1 Ile-Pro-Arg-Y PA1 Gly-Val-Arg-Y PA1 Phe-Pro-Arg-Y PA1 tosyl-Gly-Pro-Arg-Y PA1 Boc-Val-Pro-Arg-Y PA1 Bz-Gly-Pro-Arg-Y PA1 Z-Gly-Pro-Arg-Y PA1 Gly-Pro-Arg-Y PA1 (D)Phe-Val-Arg-Y PA1 (D)Val-Pip-Arg-Y PA1 (D)Tyr-Pip-Arg-Y PA1 (D)Phe-Aze-Arg-Y PA1 N-(Bz)-Phe-Val-Arg-Y PA1 (D)Val-Pro-Arg-Y PA1 (D)Val-Leu-Arg-Y PA1 (D)Pro-Phe-Lys PA1 Gly-Arg-Y PA1 CbO-Gly-Pro-Arg-Y PA1 CbO-Pro-Phe-Arg-Y PA1 CbO-Pro-Arg-Y PA1 Boc-Val-Pro-Arg-Y PA1 (D)Ala-Pro-Arg-Y PA1 (N-R)-Ala-Pro-Arg-Y PA1 (N-R)-(D)Ala-Pro-Arg-Y PA1 CbO-Sar-Pro-Arg-Y PA1 tosyl-Sar-Pro-Arg-Y PA1 Bz-Sar-Pro-Arg-Y PA1 .beta.-Ala-Pip-Arg-Y PA1 .beta.-Ala-Aze-Arg-Y PA1 .beta.-Ala-Pro-Lys-Y PA1 .beta.-Ala-Pip-Lys-Y PA1 .beta.-Ala-Pro-Orn-Y PA1 .beta.-Ala-Pip-Orn-Y PA1 .beta.-Ala-Aze-Orn-Y PA1 .alpha.-aminobutyryl-Pro-Arg-Y PA1 .alpha.-aminobutyryl-Pip-Arg-Y PA1 .alpha.-aminobutyryl-Aze-Arg-Y PA1 .alpha.-aminobutyryl-Pro-Lys-Y PA1 .alpha.-aminobutyryl-Pip-Lys-Y PA1 .alpha.-aminobutyryl-Pro-Orn-Y PA1 .alpha.-aminobutyryl-Pip-Orn-Y PA1 .alpha.-aminobutyryl-Aze-Orn-Y PA1 .alpha.-aminobutyryl-Aze-Lys-Y PA1 phenoxyacetyl-Pro-Arg-Y PA1 phenoxyacetyl-Pip-Arg-Y PA1 phenoxyacetyl-Aze-Arg-Y PA1 phenoxyacetyl-Pro-Lys-Y PA1 phenoxyacetyl-Pip-Lys-Y PA1 phenoxyacetyl-Aze-Lys-Y PA1 phenoxyacetyl-Pro-Orn-Y PA1 phenoxyacetyl-Aze-Orn-Y PA1 phenoxyacetyl-Pip-Orn-Y PA1 N-[3-R-2-(3-phenyl-2-benzamido-thio-R)-butanoyl]-Arg-Y PA1 N-[4-RO-benzenensulfonyl]-Arg-Y PA1 N-[3-R-2-(3-phenyl-2-benzamido-thio-R)-butanoyl]-N-4(RO-benzensulfonyl)-Arg -Y PA1 N-Z-Lys-Y PA1 N-Z-Orn-Y PA1 N-[3-R-3-(3-phenyl-2-benzamido-thio-R)-butanoyl]-N-Z]-Lys-Y PA1 N-[3-R-3-(3-phenyl-2-benzamido-thio-R)-butanoyl]-N-Z]-Orn-y PA1 N-[3-R-2-(3-phenyl-2-benzamido-thio-R)-butanoyl]-Lys-Y PA1 N-[3-R-2-(3-phenyl-2-benzamido-thio-R)-butanoyl]-Orn-Y .beta.-Ala-Pro-Arg-Y PA1 Bz-Ile-Glu-(.gamma. piperidyl)-Gly-Arg-Y PA1 Bz-Ile-Glu-Gly-Arg-Y PA1 Boc-Ser-Gly-Arg-Y PA1 Boc-Ile-Glu-Gly-Arg-Y PA1 Bz-Ile-Gly-Gly-Arg-Y PA1 Ile-Glu-Gly-Arg-Y PA1 Boc-Ser-(O benzoyl)-Gly-Arg-Y PA1 (D)Ile-Gly-Gly-Arg-Y PA1 Z-Leu-Gly-Arg-Y PA1 Bz-Val-Glu-Gly-Arg-Y PA1 Bz-Leu-Glu-Gly-Arg-Y PA1 Bz-Leu-Asp-Gly-Arg-Y PA1 Z-Gly-Ala-Arg-Y PA1 Phe-Glu-Gly-Arg-Y PA1 Bz-Val-Glu-Gly-Arg-Y PA1 Bz-Ile-Gln-Gly-Arg-Y PA1 Phe-Glu-Gly-Arg-Y PA1 Bz-Ile-Glu(OR)-Gly-Arg-Y PA1 Bz-Ile-Glu[OC.sub.2 H.sub.4 N(CH.sub.3).sub.2 ]-Gly-Arg-Y ##STR5## Bz-Ile-Asp(.beta. morpholinyl)-Gly-Arg-Y ##STR6## Bz-Ile-Asp(OR)Gly-Arg-Y ##STR7## (D)Ile-Glu-Gly-Arg-Y PA1 (D)Val-Leu-Lys-Y PA1 tosyl-Gly-Pro-Lys-Y PA1 Ala-Phe-Lys-Y PA1 Boc-Val-Leu-Lys-Y PA1 succinoyl-Ala-Phe-Lys-Y PA1 RO-succinoyl-Ala-Phe-Lys-Y PA1 Z-Ala-Ala-Lys-Y PA1 Z-Gly-Gly-Lys-Y PA1 Z-Phe-Phe-Lys-Y PA1 Val-Leu-Lys-Y PA1 Bz-Val-Leu-Lys-Y PA1 Gly-Pro-Lys-Y PA1 Bz-Ile-Leu-Lys-Y PA1 Ile-Leu-Lys-Y PA1 (D)-Ile-Leu-Lys-Y PA1 (D)-Ala-Leu-Lys-Y PA1 Boc-Glu-Lys-Lys-Y PA1 Bz-Leu-Leu-Lys-Y PA1 N-(6-aminohexanoyl)-Phe-Val-Arg-Y PA1 .beta.-cyclohexyl-Ala-Val-Arg-Y PA1 N-(Val)-Leu-Arg-Y PA1 N-(Bz)-Ile-Leu-Arg-Y PA1 N-(Bz)-Pro-Phe-Arg-Y PA1 N-(acetyl)-Pro-Phe-Arg-Y PA1 N-(cyclohexylcarbonyl)-Pro-Phe-Arg-Y PA1 N-(4-methyl-Bz)-Pro-Phe-Arg-Y PA1 N-(a-aminocaproyl)-Pro-Phe-Arg-Y PA1 N-(4-aminomethyl-cyclohexylcarbonyl)-Pro-Phe-Arg-Y PA1 N-(4-aminophenyl-acetyl)-Pro-Phe-Arg-Y PA1 N-(CbO)-Pro-Phe-Arg-Y PA1 N-(Bz)-Pro-.beta.-cyclohexylAla-Arg-Y PA1 N-(Bz)-Pro-Tyr-Arg-Y PA1 N-(Bz)-Pro-Phe-Gly-Arg-Y PA1 Boc-Phe-Gly-Lys-Lys-Y PA1 Boc-Phe-Glu-Lys-Y PA1 Boc-Ile-Glu-Lys-Y PA1 Z-Ala-Ala-Arg-Y PA1 Phe-Phe-Lys-Y PA1 (D)Phe-Phe-Lys-Y PA1 N-(R)-(D)-Phe-Pro-Lys-Y PA1 N-(R)-Phe-Pro-Lys-Y PA1 N-(R)-Ala-Pro-Lys-Y PA1 Sar-Pro-Lys-Y PA1 Sar-Pro-Arg-Y PA1 N-(R)-(D)-Phe-Pro-Arg-Y PA1 N-(R)-Phe-Pro-Arg-Y PA1 N-(R)-Ala-Pro-Arg-Y N.alpha.-Bz-Pro-Phe-Arg-Y PA1 N.alpha.-Bz-Pro-Phe-Arg-Y PA1 Z-Val-Val-Lys-Y PA1 Z-Leu-Leu-Lys-Y PA1 Z-Ile-Ile-Lys-Y PA1 Z-Ser-Ser-Lys-Y PA1 Z-Thr-Thr-Lys-Y PA1 Z-Cys-Cys-Lys-Y PA1 Z-Gly-Ala-Lys-Y PA1 Z-Ala-Gly-Lys-Y PA1 Z-Ala-Tyr-Lys-Y PA1 Z-Leu-Met-Lys-Y PA1 Z-Ile-Ala-Lys-Y PA1 Z-Ile-Thr-Lys-Y PA1 Z-Ser-Phe-Lys-Y PA1 Z-Cys-Val-Lys-Y PA1 Z-Met-Tyr-Lys-Y PA1 Z-Phe-Thr-Lys-Y PA1 Z-Tyr-Gly-Lys-Y PA1 Z-Tyr-Met-Lys-Y PA1 N-(CbO)-Gly-Pro-Arg-Y PA1 Gly-Pro-Arg-Y PA1 N-(phenylacetyl)-Glu-Pro-Arg-Y PA1 N-(phenylpropionyl)-Gly-Pro-Arg-Y PA1 N-(cyclohexylcarbonyl)-Gly-Pro-Arg-Y PA1 N-(capryloyl)-Gly-Pro-Arg-Y PA1 N-(benzensulfonyl)-Gly-Pro-Arg-Y PA1 N-(methanesulfonyl)-Gly-Pro-Arg-Y PA1 N-(naphthalenesulfonyl)-Gly-Pro-Arg-Y PA1 N-(isobutyloxycarbonyl)-Gly-Pro-Arg-Y PA1 N-(isobutyloxycarbonyl)-Gly-Pro-Lys-Y PA1 N-(tosyl)-Gly-Pro-Lys-Y PA1 (D)-Ser-Ser-Lys-Y PA1 (D)Thr-Thr-Lys-Y PA1 (D)Cys-Cys-Lys-Y PA1 Sar-Ala-Lys-Y PA1 (D)Ala-Gly-Lys-Y PA1 N-(R)-(D)-Ala-Gly-Lys-Y PA1 N-(R)-Ala-Gly-Lys-Y PA1 (D)-Ile-Thr-Lys-Y PA1 (D)-Ser-Phe-Lys-Y PA1 (D)-Met-Tyr-Lys-Y PA1 (D)-Phe-Thr-Lys-Y PA1 glutaryl-Lys-Lys-Y PA1 &gt;Glu-(D)Lys-Y PA1 Ala-(D)Lys-Y PA1 (D)Val-(D)Lys-Y PA1 &gt;Glu-Phe-Lys-Y PA1 (D)Val-Phe-Lys-Y PA1 Bz-(Asp).sub.4 -Lys-Y PA1 Gly-(Asp).sub.4 -Lys-Y PA1 Boc-(Asp).sub.4 -Lys-Y PA1 Bz-(Asp).sub.4 -Arg-Y PA1 (D)Asp-(Asp).sub.3 -Lys-Y PA1 (D)Asp-(Asp).sub.3 -Arg-Y PA1 Gly-(Asp).sub.4 -Arg-Y PA1 Boc-(Asp).sub.4 -Arg-Y PA1 succinoyl-Ala-Ala-Ala-Y PA1 acetyl-Ala-Ala-Ala-Y PA1 succinoyl-Ala-Pro-Ala-Y PA1 succinoyl-Ala-Ala-Pro-Val-Y PA1 succinoyl-Ala-Ala-Pro-Leu-Y PA1 acetyl-Ala-Ala-Pro-Ala-Y PA1 glutaryl-(Ala).sub.3 -Y PA1 glutaryl-(Ala).sub.2 -Y PA1 Glu-Ala-Ala-Y PA1 succinoyl(OMe)-Ala-Ala-Pro-Val-Y PA1 Boc-Ala-Y PA1 Z-Lys-Nle-Arg-Y PA1 Z-Arg-Nle-Nle-Y PA1 Bz-Arg-Val-Leu-Y PA1 Z-Arg-Val-Leu-Y PA1 Asp-Nle-Nle-Y PA1 succinoyl-Nle-Nle-Y PA1 Z-Asp-Pro-Leu-Y PA1 Z-Asp-Nle-Nle-Y PA1 Bz-Phe-Val-Arg-Y PA1 Z-Val-Lys-Lys-Y PA1 Z-Ala-Arg-Arg-Y PA1 Bz-Ala-Arg-Arg-Y PA1 Bz-Val-Lys-Lys-Y PA1 Bz-Val-Lys-Lys-Arg-Y PA1 Z-Arg-Arg-Y PA1 Z-Val-Lys-Lys-Arg-Y PA1 Z-Ala-Arg-Arg-Y PA1 Phe-Pro-Ala-Met-Y PA1 glutaryl-Gly-Phe-Y PA1 Bz-Arg-Y PA1 Bz-Arg-Gly-Leu-Y PA1 Bz-Gly-Gly-Arg-Y PA1 &lt;Glu-(D)-Phe-Pro-Phe-Phe-Y PA1 Bz-Arg-Pro-Gly-Phe-Phe-Leu-Y PA1 Bz-Arg-Pro-Gly-Phe-Phe-Pro-Y PA1 &lt;Glu-(D)Phe-Pro-Phe-Phe(D)Phe-Y PA1 &lt;Glu-(D)Phe-Pro-Phe-Phe-Val-(D)Phe-Y PA1 &lt;Glu-(D)Phe-Pro-Phe-Phe-Val-(D)Trp-Y PA1 succinoyl-Gly-Pro-Leu-Gly-Pro-Y PA1 X-Pro-Leu-Gly-Pro-(D)Arg PA1 Z-Pro-Ala-Gly-Pro-Y PA1 X-Pro-Gln-Gly-Ile-Ala-Gly-Gln-(D)Arg PA1 X-Pro-Leu-Gly-Ile-Ala-Gly-Gln-(D)Arg PA1 X-Pro-Gln-Gly-Leu-Ala-Gly-Gln-(D)Arg PA1 X-Pro-Ala-Gly-Leu-Ala-Gly-Gln-(D)Arg PA1 X-Pro-Gln-Gly-Ile-Ala-Gly-(D)Arg PA1 X-Pro-Ala-Gly-Ile-Ala-Gly-Gln-(D)Arg PA1 X-Leu-Gly-Pro-(D)Arg PA1 X-Pro-Gln-Gly-Ile-Ala-Gly-Gln-(D)Arg PA1 X-Pro-Gln-Gly-Ile-Ala-Gly-(D)Arg PA1 X-Gln-Gly-Ile-Ala-Gly-Gln-(D)Arg PA1 X-Ala-Gly-Ile-Ala-Gly-Gln-(D)Arg PA1 CbO-Pro-Ala-Gly-Pro-Y PA1 X-Pro-Pro-Gly-Ile-Ala-Gly-Gln-(D)Arg PA1 X-Pro-Leu-Gly-Ile-Ala-Gly-Arg PA1 X-Pro-Leu-Gly-Ile-Ala-Gly-(D)Arg PA1 X-Leu-Gly-Ile-Ala-Gly-Arg PA1 X-Leu-Gly-Ile-Ala-Gly-(D)Arg PA1 Gly-Pro-Leu-Gly-Y PA1 glutaryl-Gly-Arg-Y PA1 Bz-Val-Gly-Arg-Y PA1 Ile-Ile-Arg-Y PA1 Val-Gly-Arg-Y PA1 Boc-Val-Gly-Arg-Y PA1 Bz-Ile-Gly-Arg-Y PA1 Z-Gly-Gly-Arg-Y PA1 Bz-Gly-Gly-Arg-Y PA1 Gly-Gly-Arg-Y PA1 Bz-Leu-Ile-Arg-Y PA1 Bz-Leu-Leu-Arg-Y PA1 Bz-Phe-Leu-Arg-Y PA1 Pro-Gly-Arg-Y PA1 Dns-Gly-Gly-Arg-Y PA1 Dns-Glu-Gly-Arg-Y PA1 &lt;Glu-Gly-Arg-Y PA1 Bz-Leu-Gly-Arg-Y PA1 Bz-Val-Ser-Arg-Y PA1 Bz-Ile-Ser-Arg-Y PA1 Bz-Leu-Ser-Arg-Y PA1 3-Phenylpropionyl-Val-Gly-Arg-Y PA1 2-Phenylacetyl-Val-Gly-Arg-Y PA1 CbO-methylcyclohexyl-Gly-Gly-Arg-Y PA1 methylcyclohexyl-Gly-Gly-Arg-Y PA1 phenethyl-cyclohexyl-Gly-Gly-Arg-Y PA1 methylcyclohexyl-(D)Gly-Gly-Arg-Y PA1 Bz-methylcyclohexyl-(D)Gly-Gly-Arg-Y PA1 CbO-Val-Gly-Arg-Y PA1 CbO-Gly-Arg-Y PA1 CbO-Gly-(tosyl)-Arg-Y PA1 Boc-Glu-Gly-(tosyl)-Arg-Y PA1 acetyl-Glu-Gly-Arg-Y PA1 (D)Ile-Ile-Arg-Y PA1 (D)Phe-Leu-Arg-Y PA1 (D)Pro-Gly-Arg-Y PA1 (D)Leu-Gly-Arg-Y PA1 glutaryl-(Ala).sub.3 -Y PA1 succinoyl-(Ala).sub.3 -Y PA1 Z-Gly-Leu-Phe-Y PA1 glutaryl-Gly-Gly-Phe-Y PA1 Ser-Tyr-Y PA1 Pro-Arg-Y PA1 His-Ser-Y PA1 Lys-Ala-Y PA1 Arg-Ala-Y PA1 Gly-Ala-Y PA1 Asp-Ala-Y PA1 Arg-Arg-Y PA1 Gly-Pro-Y PA1 Bz-Ile-Glu(.gamma. OR)-Gly-Arg-Y PA1 Tosyl-Ile-Glu-Gly-Arg-Y PA1 (D)Val-Leu-Gly-Arg-Y PA1 Boc-Val-Leu-Gly-Arg-Y PA1 Boc-Leu-Gly-Arg-Y PA1 Bz-Ile-Glu-Gly-Arg-Y PA1 Glu-Gly-Arg-Y PA1 Bz-Val-Gly-Arg-Y PA1 Boc-Val-Ser-Gly-Arg-Y PA1 Boc-Ser-Gly-Arg-Y PA1 (D)Ser-Gly-Arg-Y PA1 Bz-Val-Leu-Gly-Arg-Y PA1 Z-Val-Leu-Gly-Arg-Y PA1 (D)Val-Ser-Gly-Val-Ser-Gly-Arg PA1 Boc-Ile-Glu-Gly-Arg-Y PA1 glutaryl-Gly-Arg-Y PA1 Boc-Val-Ser-Gly-Arg-Y PA1 Boc-Ser-Gly-Arg-Y PA1 Boc-Val-Leu-Gly-Arg-Y PA1 Boc-Ser(O benzyl)-Gly-Arg-Y PA1 Val-Gly-Arg-Y PA1 &lt;Glu-Gly-Arg-Y PA1 Bz-Val-Gly-Arg-Y PA1 (D)Val-Gly-Arg-Y PA1 (D)&lt;Glu-Gly-Arg-Y PA1 (D)Glu-Gly-Arg-Y PA1 Z-Gly-Pro-Y PA1 Bz-Gly-Pro-Y PA1 succinoyl-Gly-Pro-Y PA1 X-Gly-Pro PA1 X-Arg-Pro PA1 X-Ala-Pro PA1 X-Lys-Pro PA1 X-Glu-Pro PA1 X-Asp-Pro PA1 X-His-Pro PA1 X-Leu-Pro PA1 X-Ile-Pro PA1 X-Tyr-Pro PA1 X-Trp-Pro PA1 X-Phe-Pro PA1 X-Val-Pro PA1 X-Gln-Pro PA1 X-Asn-Pro PA1 X-Ser-Pro PA1 X-Cys-Pro PA1 X-Thr-Pro ##STR8## acyl-Arg-Pro-Y acyl-Ala-Pro-Y PA1 acyl-Lys-Pro-Y PA1 acyl-Glu-Pro-Y PA1 acyl-Asp-Pro-Y PA1 acyl-His-Pro-Y PA1 acyl-Leu-Pro-Y PA1 acyl-Ile-Pro-Y PA1 acyl-Tyr-Pro-Y PA1 acyl-Trp-Pro-Y PA1 acyl-Phe-Pro-Y PA1 acyl-Val-Pro-Y PA1 acyl-Gln-Pro-Y PA1 acyl-Asn-Pro-Y PA1 acyl-Ser-Pro-Y PA1 acyl-Cys-Pro-Y PA1 acyl-Thr-Pro-Y PA1 Gly-Pro-Y PA1 Arg-Pro-Y PA1 Ala-Pro-Y PA1 Lys-Pro-Y PA1 Glu-Pro-Y PA1 Asp-Pro-Y PA1 His-Pro-Y PA1 Leu-Pro-Y PA1 Ile-Pro-Y PA1 Tyr-Pro-Y PA1 Trp-Pro-Y PA1 Phe-Pro-Y PA1 Val-Pro-Y PA1 Gln-Pro-Y PA1 Asn-Pro-Y PA1 Ser-Pro-Y PA1 Cys-Pro-Y PA1 Thr-Pro-Y PA1 (D)Val-Leu-Arg-Y PA1 (D)Pro-Phe-Arg-Y PA1 Z-Phe-Arg-Y PA1 Leu-Met-Lys-Y PA1 Ser-Leu-Met-Y PA1 Asp-Trp-Arg-Y PA1 Phe-Arg-Y PA1 Z-Pro-Phe-Arg-Y PA1 Val-Leu-Arg-Y PA1 Ile-Leu-Arg-Y PA1 (D)Ile-leu-Arg-Y PA1 acetyl Pro-Phe-Arg-Y PA1 Bz-Pro-Phe-Arg-Y PA1 cyclopentylcarbonyl-Pro-Phe-Arg-Y PA1 acetyl-Phe-Arg-Y PA1 Z-Leu-Gly-Arg-Y PA1 N-(caproyl)-Pro-Phe-Arg-Y PA1 N-(3-phenylpropanoyl)-Pro-Phe-Arg-Y PA1 N-(tosyl)-Pro-Phe-Arg-Y PA1 N-(Boc)-Leu-Gly-Arg-Y PA1 CbO-Phe-Arg-Y PA1 Phe-Arg-Y PA1 X-Phe-Ser-Pro PA1 X-Gly-Gly-Gly PA1 X-Gly-His-Leu PA1 X-Phe-His-Leu PA1 X-Pro-Phe-Arg PA1 X-Phe-Ala-Pro PA1 X-(D)Phe-Ala-Pro PA1 X-(D)Phe-His-Leu PA1 X-(D)Phe-Ser-Pro PA1 Met-Phe-Gly-Y PA1 Leu-Phe-Gly-Y PA1 X-Phe-Phe-Ala PA1 X-Phe-O-Phe-Ala PA1 X-Gly-O-Gly-Phe PA1 X-Gly-O-Phe-Phe PA1 X-Gly-O-Leu-Ala PA1 X-Gly-Gly-Phe PA1 cystinoyl-Y PA1 R-Gly-Leu-Y PA1 R-Ala-Leu-Y PA1 R-Gly-Phe-Y PA1 furylacryloyl-Gly-Leu-Y PA1 furylacryloyl-Gly-Phe-Y PA1 thienylacryloyl-Gly-Leu-Y PA1 thienylacryloyl-Ala-Leu-Y PA1 thienylacryloyl-Gly-Phe-Y PA1 &lt;Glu-His-Pro-Y PA1 &lt;Glu-(benzimidazolyl)His-Pro-Y
All amino acids are in the L-form unless otherwise indicated. The enzyme nomenclature developed by the Enzyme Committee of the International Union of Biochemistry is used herein.
Recently, many synthetic peptide substrates have been prepared for different proteolytic enzymes. These substrates have generally included a chromogenic or fluorogenic group for measuring the amount of substrate hydrolysis by a particular enzyme or class of enzymes. For example, U.S. Pat. No. 3,886,136 discloses several chromogenic substrates for assaying enzymes of the class E.C. 3.4.4 (now class E.C. 3.4.21). Examples of this class include trypsin (E.C. 3.4.21.4), chymotrypsin (E.C. 3.4.21.1), plasmin (E.C. 3.4.21.7) and thrombin (E.C. 3.4. 21.5), among others. The substrates disclosed in this patent have the general formula: ##STR2## wherein R.sub.3 and R.sub.4 are alkyl groups having 3-8 carbons, R.sub.4 can also be benzyl or phenyl, R.sub.5 is hydrogen or ##STR3## n is 2,3 or 4, --NH--R.sub.6 is the chromogenic group, X is CH.sub.2 or a single bond, and R.sub.1 and R.sub.2 can be selected from a variety of groups which is not critical to this discussion. U.S. Pat. No. 4,016,042 discloses chromogenic or fluorogenic substrates for proteolytic enzymes of the class E.C. 3.4.21. These substrates are derivatives of Pro-X-Y-R where X is Phe, Tyr, phenylglycine or .beta.-cyclohexylalanine, Y is Arg or Lys and R is the chromogenic or fluorogenic group.
U.S. Pat. No. 4,061,625 discloses several chromogenic substrates useful for assaying thrombin (E.C. 3.4.21.5) or thrombin-like enzymes. These substrates have the formula (D) A.sub.1 -A.sub.2 -Arg-R where A.sub.1 is Phe or Tyr, A.sub.2 is Aze, Pro or Pip, and R is the chromogenic group. In these substrates the symbol (D) A.sub.1 signifies that A.sub.1 is in the D form but A.sub.2 and Arg are in the L-form. This symbolism is used throughout this application.
U.S. Pat. No. 4,070,245 discloses several chromogenic or fluorogenic substrates useful for assaying enzymes of the class E.C. 3.4.21. These substrates are derivatives of Gly-Pro-X-R where X is Lys or Arg and R is a chromogenic or fluorogenic group.
U.S. Pat. No. 4,137,225 discloses chromogenic substrates for proteases (serine proteases) of the class E.C. 3.4.21. This patent discloses substrates of the formula (D)A.sub.1 -A.sub.2 -A.sub.3 -R where A.sub.1 and A.sub.2 are selected from the group of amino acids Gly, Ala, Val, Leu, Ile, Pip, Pro or Aze, A.sub.2 can also be Phe, A.sub.3 is Arg, Lys or Orn and R is the chromogenic group.
U.S. Pat. No. 4,147,692 discloses a fluorogenic substrate, Gly-Pro-R where R is the fluorogenic group, for assaying the enzyme X-prolyl dipeptidyl aminopeptidase. U.S. Pat. No. 4,119,620 also discloses substrates for the enzyme X-prolyl dipeptidyl aminopeptidase. These substrates have the general formula X-Pro-Y wherein X can be any amino acid and Y is a chromogenic group.
U.S. Pat. No. 4,188,264 discloses several chromogenic or fluorogenic substrates for the clotting enzyme of horseshoe crabs. These substrates have the general formula R.sub.1 -Gly-Arg-R.sub.2 wherein R.sub.1 is selected from the group comprising N-protected L-amino acids, N-protected L-peptides, (D)amino acid-L-amino acid or (D) amino acid-L-peptide and R.sub.2 is the chromogenic or fluorogenic group.
U.S. Pat. No. 4,028,318 discloses chromogenic substrates for serine proteases (E.C. 3.4.21.) especially factor Xa (E.C. 3.4.21.6). These substrates have the general formula R-A.sub.1 -A.sub.2 -Gly-Arg-R.sub.1 where A.sub.1 may be a single bond or Gly, Ala, Val, Leu, Ile, Pro, Met, Phe or Tyr, A.sub.2 may be Glu, Gln, Asp or Asn, R is H or a blocking group and R.sub.2 is the chromogenic group.
U.S. Pat. No. 4,056,519 discloses fluorogenic substrates for the enzyme, plasmin (E.C. 3.4.21.7.). These substrates have the general formula ##STR4## wherein R.sub.1 is benyloxycarbonyl, R.sub.4 is 4-methoxy-2-naphthylamine (MNA), R.sub.2 and R.sub.3 may be H, alkyl, hydroxyalkyl, mercaptoalkyl, methylthioalkyl, benzyl or hydroxybenzyl. The preferred substrates are Z-Gly-Gly-Lys-MNA or Z-Ala-Ala-Lys-MNA.
Other U.S. patents which disclose chromogenic or fluorogenic peptide substrates include the following: Nos. 3,144,484 for trypsin (E.C. 3.4.21.4); 3,536,588 for Leu aminopeptidase (E.C. 3.4.11.1); 3,591,459 for amino acid arylamidase; 3,607,859 for neutral protease (microbial metalloenzymes, E.C. 3.4.23.4); 3,703,441; 3,769,173; 3,773,626; 3,892,631 and 4,177,109 for .gamma. Glu transpeptidase (E.C. 2.3.2.2); 3,745,212 for pancreatic endopeptidases; 3,884,896, 4,191,808 and 4,191,809 for peptide peptidohydrolases such as class E.C. 3.4.21; 4,046,633 for renin (E.C. 3.4.99.19); 4,108,726 and 4,115,374 for angiotensin converting enzyme (peptidyldipeptide hydrolase, E.C. 3.4.15.1); 4,116,774 for Leu aminopeptidase, Cys aminopeptidase (E.C. 3.4.11.3) and .gamma.-Glu transpeptidase; 4,138,394 for collagenase (E.C. 3.4.24.3); and 4,207,232 for factor Xa (E.C. 3.4.21.6). U.S. Pat. Nos. 3,862,011; 4,155,916 and 4,167,449 contain tables listing specific chromogenic or fluorogenic substrates for various proteolytic enzymes. Still further colorometric substances useful for assaying one or more of thrombin, horesehoe crab coagulating enzyme, urokinase or Factor XIIA are shown in U.S. Pat. No. 4,215,047. Further substrates for thrombin and trypsin-like enzymes are shown in U.S. Pat. Nos. 4,217,269, 4,219,497 and 4,221,706. U.S. Pat. No. 4,216,142 describes substrates especially useful with the enzyme inhibitor antithrombin III.
In addition to the above-identified U.S. patents, chromogenic or fluorogenic peptide substrates for various proteolytic enzymes have also been described in many literature articles. Representative articles which refer to enzymes not previously discussed herein include the following: Yoshimota et al., Biochim. Biophys. Acta 569, 184 (1979) for post-proline cleaving enzyme (E.C. 3.4.21.-) using Z-Gly-Pro-R.sub.1 wherein R.sub.1 is a chromogenic or fluorogenic group as the substrate; Grant et al., Biochim. Biophys. Acta 567, 207 (1979) for enterokinase (E.C. 3.4.21.9) using Gly-(Asp).sub.4 -Lys-naphthylamide; Reilly et al., Biochim. Biophys. Acta 621, 147 (1980) for elastase (E.C. 3.4.21.11) using succinyl-(Ala).sub.3 -p-nitroanilide; Pozgay et al, Eur. J. Biochem 95, 115 (1979) for Subtilisin Carlsberg (E.C. 3.4.21.14) using, for example, Z-Arg-(Nle).sub.2 -p-nitroanilide; and, Lojda, Histochem. 64, 205 (1979) for brush border endopeptidase using glutaryl-(Ala).sub.3 -MNA or succinyl-(Ala).sub.3 -l-naphthylamide.
There are many disadvantages in the use of the chromogenic and fluorogenic substrates of the prior art for the assay of mammalian enzymes, especially proteolytic and peptidase enzymes. The chromogenic assays frequently use a substrate that reacts with a given enzyme to release p-NO.sub.2 -aniline as the chromophore. p-NO.sub.2 -aniline has a low molar extinction coefficient, thus a high concentration of substrate is required in order to permit formation of sufficient product for accurate and precise measurement. This requirement for high substrate concentration is disadvantageous; the substrates are often very expensive to make. In addition, solubility of these substances in buffer is often low. Some of the chromogenic substrates (e.g. benzoyl-Phe-Val-Arg-p-NO.sub.2 -anilide and (D)Phe-Pip-Arg-p-NO.sub.2 -anilide) cannot be dissolved in their reaction buffers at concentrations high enough to allow the enzyme-substrate reaction to proceed under conditions of zero order reaction kinetics (i.e. under conditions wherein a substantial excess of substrate is present). See Lehninger, A., Biochemistry (1970) p. 153 et seq. In consequence, some of the chromogenic substrates must be used at concentrations within the range of mixed first order and zero order enzyme kinetics. Use of these substrates at concentrations appropriate to first order kinetics is often infeasible because the color produced is often so weak that it cannot be detected, even instrumentally, unless the sample is colorless. Even at the concentrations used, it is often necessary to employ special techniques, e.g. sonication, heating at 50.degree. C., etc. to effect dissolution of the p-nitroanilide containing substrate.
There are still other disadvantages of the p-nitroaniline-labeled chromogenic substrates. The amide bond formed between an amino acid residue of a given substrate and p-NO.sub.2 -aniline to yield the corresponding p-NO.sub.2 -anilide is relatively unstable and can undergo spontaneous hydrolysis at pH 9 and higher pH's. The spontaneous hydrolysis is particularly disadvantageous for the assay, e.g., of human glandular kallikreins, and other enzymes having pH optima within the range of pH 9 to 10. Still a further problem with this type of substrate is that the chromophoric reaction product, p-NO.sub.2 -aniline, is not stable and is readily oxidized. Thus, the measurement must be made immediately or accuracy is lost.
A further disadvantage of the p-nitroanilide substrates is that they are not stable under ordinary ambient conditions and are especially prone to oxidative degradation. As a result, special storage conditions are needed. Moreover, the instability problems are such that precise repetition of substrate in different batch preparations is difficult to achieve and may often not occur. This in its turn causes quality control problems in the marketplace. Even using the same batch, moreover, repetitive analyses conducted days or weeks apart may field varying results due to instability and interim degradation of the substrate.
Others of the chromogenic substrates (e.g., benzoyl-Pro-Phe-Arg-p-NO.sub.2 -anilide) have been thought to be unreactive with a given enzyme (e.g., the last-mentioned substrate with human urinary kallikrein), whereas in fact the substrates have a high affinity (low K.sub.m) for their designated enzymes and a relatively low maximum velocity of reaction (V.sub.max). Because of the low V.sub.max, insufficient chromophore is generated to allow its measurement. Not uncommonly, the substrates of high affinity and low V.sub.max also inhibit the activity of the enzyme to be measured (so-called substrate inhibition) at substrate concentrations only slightly greater than K.sub.m. Thus, Chung et al, Adv. Experimental Med. and Biol., 120A, 115-125 (1979) have shown that the K.sub.m of reaction of human urinary kallikrein with Pro-Phe-Arg-[.sup.3 H]benzylamide is 3 .mu.M, and substrate inhibition is manifest at 5 .mu.M.
The sensitivity of chromogenic substrates for proteolytic and peptidase enzymes is often low. In consequence, it may be difficult or impossible to assay an enzyme present in minute quantity. As for other assays of enzymic activity, sensitivity is in large part a function of the ratio of V.sub.max /K.sub.m and the detectability of the product. As noted above, solubility of the substrate may compromise sensitivity of an assay when a sufficient substrate concentration cannot be obtained to allow velocity of the reaction to approximate V.sub.max. Since the detectability of the chromophoric product is in general poor, it is often necessary to use a substrate of relatively low affinity (high K.sub.m) and high V.sub.max in order to develop a practical chromogenic assay. Typically, substrates of high affinity provide the greatest specificity or selectivity of reaction. Hence, some chromogenic assays must use substrates of less than optimum specificity for their designated enzymes in order to allow for the generation of sufficient chromophore for accurate measurement.
Chromophores other than p-NO.sub.2 -aniline can, in principle, be used. However, the substitution of one leaving group for another often results unpredictably and for unknown reasons, in sensitivity, specificity and other differences. Chromophoric substitution may make the resulting substrate more or less sensitive or more or less specific than the corresponding p-NO.sub.2 -anilide substrate. For example, we have found that Pro-Phe-Arg-anilide has a lower K.sub.m and a higher V.sub.max on reaction with human urinary kallikrein than does Pro-Phe-Arg-benzylamide. Further, neither aniline nor benzylamine is a strong chromophore; both are far more difficult to detect colorimetrically than p-NO.sub.2 -aniline. Even more marked changes have been encountered. For example, benzoyl-Val-Gly-Arg-p-NO.sub.2 -anilide and &lt;Glu-Gly-Arg-p-NO.sub.2 -anilide are reactive with human urokinase, but the corresponding benzylamides are not reactive with urokinase and are highly reactive with the clotting enzyme of the horseshoe crab.
Some chromophores, e.g., .beta.-naphthylamine, are more readily detected than is p-NO.sub.2 -aniline but may be carcinogenic. Other chromophores, e.g., p-NO.sub.2 -phenol, are as difficultly detected as is p-NO.sub.2 -aniline. These chromophores are incorporated into a given substrate via an ester linkage and have the expected advantages and disadvantages. Typically, the reaction of a p-NO.sub.2 -phenyl ester with a given enzyme has a significantly higher V.sub.max than does the reaction using the same enzyme and the corresponding p-NO.sub.2 -anilide substrate. However, spontaneous hydrolysis of the ester substrate becomes serious at pH levels above 7.0. Thioester chromogenic substrates have similar advantages and disadvantages. Both ester and thioester substrates may under some conditions behave as active esters and hence irreversibly inhibit the enzyme to be assayed.
Additional known chromophores, such as hydroxycoumarins and methylcoumarins, share the solubility, affinity, detectability, cost, and other disadvantages of p-nitroaniline, albeit not necessarily to the same degree when otherwise analogous substrates are compared.
Fluorogenic substrates have many of the same disadvantages as the chromogenic substrates. Fluorogenic substrates generally use a derivative of naphthylamine in either amide or ester linkage. More recently, substrates have been designed to contain a coumarin derivative as the fluorogenic moiety. As noted above, the naphthylamine derivatives may be carcinogenic. Apparently, the substrates and their fluorescent products are not stable as judged by the manufacturers' recommended storage precautions for these compounds. Fluorogenic substrates as a class are even less soluble than the corresponding p-NO.sub.2 -anilides, and it is frequently necessary first to dissolve a given fluorogenic substrate in an organic solvent such as DMSO (dimethylsulfoxide) before adding the substrate to a buffered aqueous solution compatible with the enzyme-substrate reaction. The organic solvent may damage or partially denature the enzyme to be measured. Even if the denaturation of enzyme is slight, extensive controls must be inserted in the assay protocol in order to retain adequate precision and accuracy. Some fluorogenic substrates are not highly soluble even in an organic solvent. Thus, the assistance of the organic solvent may not suffice to yield a substrate concentration in the reaction mixture adequate to support a reaction obeying zero order enzyme kinetics. Consequently, many fluorogenic assays use substrate concentrations between the ranges of first order and zero order enzyme kinetics. The fluorophoric leaving groups used at present are even less soluble in aqueous solution than their parent substrates and may tend to precipitate as the enzyme-substrate reaction progresses. A disadvantage unique to fluoroscence assays is self-quenching of fluoroscence. Further intrinsic fluoroscence of some biological fluids may equal or exceed in intensity that of the fluorophor released from a given substrate. The intensity of the intrinisic fluoroscence of biological specimen varies widely; hence, any specimen may differ markedly from another, even of the same type.
Information on aspartyl-.sup.3 [H]-benzylamide as a substrate for angiotensin aminopeptidase and lysine-.sup.3 [H]-benzylamide as a substrate for kinin, converting enzyme (a carboxypeptidase enzyme) was made publicly available through National Institutes of Health channels, on behalf of the present inventors in 1979. This work was preliminary to the present invention. A pertinent publication emanating from this laboratory and describing substrate work of these inventors within the scope of this invention is Ryan, W. and Ryan, U.S., "Biochemical and Morphological Aspects of the Actions and Inactivation of Kinins and Angiotensins" contained in Enzymatic Release of Vasoactive Peptides, edited by F. Gross and G. Vogel, published mid-1980. The article additionally makes reference to tritiated benzoyl derivatives of the tripeptides Gly-Gly-Gly, Gly-His-Leu, Pro-Phe-Arg, Phe-His-Leu and Phe-Ala-Pro as angiotensin converting enzyme substrates and to the compound Pro-Phe-Arg-.sup.3 [H] benzylamide as a glandular kallikrein substrate.
The present invention obviates the disadvantages of the prior art colorometric and fluorometric substrates by providing a class of radiolabelled compounds, each member of which is useful as an assay substrate for one or more proteolytic or peptidase enzymes. Each of the compounds of the invention is a radiolabelled benzoyl, p-OH-phenyl-propanoyl or benzylamide derivative of an amino acid or oligopeptide. In each instance, the benzoyl, p-OH-phenyl-propanoyl or benzylamide moiety contains the radiolabel. The radiolabelled moieties allow the highly sensitive detection of a given substrate or one of the products formed by enzymic hydrolysis, or both. Thus, enzymic activity can be measured at high sensitivity in terms of the rate of substrate utilization and/or product formation. In addition, the radiolabelled substrates and their cleavage products are stable compounds. The substrates, properly stored, retain activity for at least about two years. They can be used at concentrations in the range of first order enzyme kinetics so that concentrations as low as 50 nM are conventional and solubility in test fluids is never a problem. In addition, since 25 mg. of a given radiolabelled substrate of this invention will theoretically provide about five million tests whereas the same quantity of present commercial colorimetric substrates, e.g. from Kabi AB or Pentapharm, would yield theoretically, about 500 tests and the commercial fluorogenic substrates from e.g. American Hospital Supply, Dade Reagents Division, would theoretically yield also about 500 tests, it is evident that the cost savings available according to this invention are significant.
The amino acid or oligopeptide present in each substrate of this invention was originally selected to favor the affinity and selectivity of binding of that substrate for a designated enzyme. Thus, a substrate such as tosyl-Gly-Pro-Arg-[.sup.3 H]benzylamide incorporates a tripeptide sequence of a known natural substrate for thrombin. In similar vein, [.sup.3 H]benzoyl-Phe-Arg was designed for carboxypeptidase B and related enzymes and &lt;Glu-Gly-Arg-[.sup.3 H]benzylamide was designed for urokinase. It has unexpectedly been found, however, that the radiolabelled moieties and even the radiolabels per se may enhance or decrease the affinity, selectivity and even specificity of a given substrate for its designated enzyme. For example, &lt;Glu-Gly-Arg-[.sup.3 H]benzylamide was found to be unreactive with urokinase, the enzyme for which the analogous colorimetric substrate is specific, but was highly reactive with the horseshoe crab clotting enzyme. Conversely, (D)Phe-Pro-Arg-[.sup.3 H]benzylamide was found to have a higher affinity (lower K.sub.m) for thrombin than that reported for the corresponding p-NO.sub.2 -anilide. Similarly, it has unexpectedly been found that substitutions or additions distant from the cleavage site of the substrate may unpredictably influence the kinetic behavior of a given substrate. For example, the addition of a biotinyl group to (D)Phe-Pro-Arg-[.sup.3 H]benzylamide to yield biotinyl-(D)Phe-Pro-Arg-[.sup.3 H]benzylamide yields a substrate for thrombin less than half as reactive as (D)Phe-Pro-Arg-[.sup.3 H]benzylamide itself.
The substrates of the present invention may be utilized in concentrations well within the range of first order enzyme kinetics. These substrates, labelled at high specific radioactivity, can be used in nanomolar or sub-nanomolar quantities without compromising the detection of substrate utilization or product formation. The typical K.sub.m for reaction of these substrates with their designated enzymes ranges from 1 .mu.M to 1 mM. Because of the ready detectability of each substrate and at least one of its cleavage products, the substrates of this invention can be used at concentrations below K.sub.m and well within the range of first order enzyme kinetics. Within the latter range, enzyme activity, expressed as percent utilization of substrate per unit time, becomes independent of the actual substrate concentration; and the resulting assay yields results both more accurate and precise than those conducted with substrate at a concentration within the range of mixed first and zero order enzyme kinetics. Other advantages have been found. Because low concentrations of substrate can readily be used, solubility of substrate poses no problem, and organic solvents are not needed. Further, substrate inhibition and product inhibition are far less likely to occur. On the other hand, these substrates can be used at concentrations greater than the range of first order kinetics, either by raising the concentration of the radiolabelled substrate itself or by adding the corresponding non-radiolabelled compound.